Neurochemical characterization and sexual dimorphism of projections from the brain to abdominal and subcutaneous white adipose tissue in the rat

Abstract

Retroperitoneal white adipose tissue (rWAT) and subcutaneous (inguinal) white adipose tissue (iWAT) are both innervated and regulated by sympathetic efferents, but the distribution and identity of the cells in the brain that regulate sympathetic outflow are poorly characterized. Our aim was to use two isogenic strains of a neurotropic virus (pseudorabies, Bartha) tagged with either green or red fluorescent reporters to identify cells in the brain that project to rWAT and/or iWAT. These viruses were injected into separate WAT depots in male and female Sprague Dawley rats. Retrogradely labeled neurons in the CNS were characterized by immunohistochemistry and PCR. For the latter, laser capture of individual virally labeled neurons was used. All virally labeled brain regions contained neurons projecting to either and both WAT depots. Neurons to abdominal fat were the most abundant in males, whereas females contained a greater proportion of neurons to subcutaneous via private lines and collateral branches. Retrogradely labeled neurons directed to WAT expressed estrogen receptor-α (ERα), and fewer neurons to subcutaneous WAT expressed ERα in males. Regardless of sex, projections from the arcuate nucleus were predominantly from pro-opiomelanocortin cells, with a notable lack of projections from agouti-related protein-expressing neurons. Within the lateral hypothalamus, neurons directed to rWAT and iWAT expressed orexin and melanin-concentrating hormone (MCH), but male rats had a predominance of MCH directed to iWAT. In conclusion, the neurochemical substrates that project through polysynaptic pathways to iWAT and rWAT are different in male and female rats, suggesting that metabolic regulation of rWAT and iWAT is sexually dimorphic.

Figure 1.

Photograph (A) and schematics (C, E, G, I) (adapted from Paxinos and Watson (1998) illustrate the areas (boxed regions) and bregma levels shown in the photomicrographs on the right. B, D, F, H, J, Photomicrographs of neurons projecting polysynaptically to abdominal (green), subcutaneous (red), and both (yellow; white arrows) WAT depots in the spinal cord (B; between T8 and T10), raphe magnus (D), paraventricular nucleus (F), medial preoptic region (H), and arcuate nucleus (J) of rats allowed to survive for 3 d (B) or 5 d (D, F, H, J). 3V, Third ventricle; gm, gray matter; L, lumbar segment of the spinal cord; ox, optic chiasm; py, pyramidal tract; T, thoracic segment of the spinal cord; wm, white matter. Scale bars: 100 μm.

Figure 2.

The relative proportion of neurons projecting to abdominal, subcutaneous, and both fat depots in the forebrain and hindbrain of both male (n = 5) and female (n = 4) rats injected into the right retroperitoneal and inguinal WAT and allowed to survive for 5 d. Both depots, Neurons projecting to both fat depots; DLPAG, dorsolateral PAG; DMPAG, dorsomedial PAG; LPAG, lateral PAG; VLPAG, ventrolateral PAG. *p < 0.05 compared to male neurons projecting to the subcutaneous fat depot; ^#p < 0.05 compared to female neurons projecting to the subcutaneous fat depot; ⁺p < 0.05 compared to male neurons projecting to the abdominal fat depot; ^†p < 0.05 compared to female neurons projecting to the abdominal fat depot.

Figure 3.

The percentage of PRV-positive neurons projecting to abdominal or subcutaneous WAT depots that also contained estrogen receptor-α in the forebrain and brainstem of male (n = 11) and female (n = 15) rats allowed to survive for 5 d. *p < 0.05 compared to male neurons projecting to the subcutaneous fat depot.

Figure 4.

A–F, Photomicrographs illustrating the colocalization of agouti-related protein (D, F; blue) or γ-melanocyte stimulating hormone (A, C; blue) and neurons projecting polysynaptically to abdominal (B, C, E, F; green), subcutaneous (B, C, E, F; red), or both WAT depots (B, C, E, F; yellow) in rats allowed to survive for 5 d and treated with colchicine for 16 h. PRV-positive neurons projecting to subcutaneous and/or abdominal WAT were not colocalized with AgRP-positive neurons in the arcuate (F), but neurons projecting to WAT depots were found to contain γ-MSH (top insets). Scale bar: (in F) A–F, 200 μm; inset, 10 μm.

Figure 5.

Photomicrographs and bar graphs illustrating the colocalization of MCH (left; green) or orexin-A (right; green) and neurons projecting polysynaptically to WAT depots (left and right; red) in rats (n = 6 females; n = 8 males) allowed to survive for 5 d. PRV-positive neurons projecting to WAT were colocalized with both MCH and orexin-A in the perifornical region (top; insets) and lateral hypothalamus (bottom; insets). fx, Fornix; ic, internal capsule. Scale bar: 200 μm; inset, 20 μm. ⁺p = 0.05 compared to male neurons projecting to the abdominal fat depot; ^$p = 0.051 compared to male neurons projecting to the abdominal fat depot.